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Do Osmolytes Impact the Structure and Dynamics of Myoglobin?

ABSTRACT: Osmolytes are small organic compounds that can affect the stability of proteins in living cells. The mechanism of osmolytes' protective effects on protein structure and dynamics has not been fully explained, but in general, two possibilities have been suggested and examined: a direct interaction of osmolytes with proteins (water replacement hypothesis), and an indirect interaction (vitrification hypothesis). Here, to investigate these two possible mechanisms, we studied myoglobin-osmolyte systems using FTIR, UV-vis, CD, and femtosecond IR pump-probe spectroscopy. Interestingly, noticeable changes are observed in both the lifetime of the CO stretch of CO-bound myoglobin and the spectra of UV-vis, CD, and FTIR upon addition of the osmolytes. In addition, the temperature-dependent CD studies reveal that the protein's thermal stability depends on molecular structure, hydrogen-bonding ability, and size of osmolytes. We anticipate that the present experimental results provide important clues about the complicated and intricate mechanism of osmolyte effects on protein structure and dynamics in a crowded cellular environment.

SUBMITTER: Kossowska D

PROVIDER: S-EPMC6321238 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Do Osmolytes Impact the Structure and Dynamics of Myoglobin?

Kossowska Dorota D Kwak Kyungwon K Cho Minhaeng M

Molecules (Basel, Switzerland) 20181203 12

Osmolytes are small organic compounds that can affect the stability of proteins in living cells. The mechanism of osmolytes' protective effects on protein structure and dynamics has not been fully explained, but in general, two possibilities have been suggested and examined: a direct interaction of osmolytes with proteins (water replacement hypothesis), and an indirect interaction (vitrification hypothesis). Here, to investigate these two possible mechanisms, we studied myoglobin-osmolyte system ...[more]

PMID: 30513982

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Project description:BACKGROUND: Present in various species, the knottins (also referred to as inhibitor cystine knots) constitute a group of extremely stable miniproteins with a plethora of biological activities. Owing to their small size and their high stability, knottins are considered as excellent leads or scaffolds in drug design. Two knottin families contain macrocyclic compounds, namely the cyclotides and the squash inhibitors. The cyclotide family nearly exclusively contains head-to-tail cyclized members. On the other hand, the squash family predominantly contains linear members. Head-to-tail cyclization is intuitively expected to improve bioactivities by increasing stability and lowering flexibility as well as sensitivity to proteolytic attack. RESULTS: In this paper, we report data on solution structure, thermal stability, and flexibility as inferred from NMR experiments and molecular dynamics simulations of a linear squash inhibitor EETI-II, a circular squash inhibitor MCoTI-II, and a linear analog lin-MCoTI. Strikingly, the head-to-tail linker in cyclic MCoTI-II is by far the most flexible region of all three compounds. Moreover, we show that cyclic and linear squash inhibitors do not display large differences in structure or flexibility in standard conditions, raising the question as to why few squash inhibitors have evolved into cyclic compounds. The simulations revealed however that the cyclization increases resistance to high temperatures by limiting structure unfolding. CONCLUSION: In this work, we show that, in contrast to what could have been intuitively expected, cyclization of squash inhibitors does not provide clear stability or flexibility modification. Overall, our results suggest that, for squash inhibitors in standard conditions, the circularization impact might come from incorporation of an additional loop sequence, that can contribute to the miniprotein specificity and affinity, rather than from an increase in conformational rigidity or protein stability. Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design.

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Do Osmolytes Impact the Structure and Dynamics of Myoglobin?

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Do Osmolytes Impact the Structure and Dynamics of Myoglobin?

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